Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients, and drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular calcification, including stimulation of osteo/chondrogenic differentiation, vesicle release, apoptosis, loss of inhibitors, and ECM matrix degradation. These studies suggest a major role for elevated P in promoting osteo/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic providing a major stimulus for vascular calcification in CKD. Unravelling the complex regulatory pathways that mediate the effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive effects of vascular calcification in CKD patients.

Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. Objective: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Methods and Results: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. Conclusions: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Objectives: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. Methods and Results: Transmission electron microscopy showed that both nonmineralized and mineralizedMVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. Conclusions:In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes. (Circ Res. 2011;109:e1-e12.) Key Words: matrix vesicles Ⅲ annexin Ⅲ calcification Ⅲ vascular smooth muscle cells Ⅲ calcium Ⅲ proteomics V ascular calcification is the deposition of apatite mineral in the medial or intimal layers of the vessel wall and is a clinically significant pathology in atherosclerosis, diabetes, chronic kidney disease, and aging. Once established, vascular calcification is progressive, particularly in association with raised levels of extracellular mineral ions such as calcium and phosphate. 1 Recent nuclear magnetic resonance studies have shown that the structural organization of the molecular components of vascular mineralizations are identical to those in bone. 2,3 This implies mechanistic similarities during the earliest phases of initiation of mineral nucleation in both tissues.During developmental osteogenesis/chondrogenesis, specialized membrane-bound bodies called matrix vesicles (MVs), which originate from the plasma membrane of chondrocytes and osteoblasts, serve as nucleation sites for hydroxyapatite. 4 In cartilage, MV production occurs throughout the growth plate, but MVs are "mineralization competent" only in the hypertrophic zone. 4 This transition is induced by an intracellular calcium signal that initiates changes in gene transcription and the subsequent release of MVs that are able to nucleate mineral to form hydroxyapatite nanocrystals. 5 Mineralization-competent MVs are enriched with the calcium-binding annexins (Anx) A2, A5, and A6 and surface Original received December 8, 2010; revisi...

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

DYT1 dystonia is a severe form of young-onset dystonia caused by a mutation in the gene that encodes for the protein torsinA, which is thought to play a role in protein transport and degradation. We describe, for the first time to our knowledge, perinuclear inclusion bodies in the midbrain reticular formation and periaqueductal gray in four clinically documented and genetically confirmed DYT1 patients but not in controls. The inclusions were located within cholinergic and other neurons in the pedunculopontine nucleus, cuneiform nucleus, and griseum centrale mesencephali and stained positively for ubiquitin, torsinA, and the nuclear envelope protein lamin A/C. No evidence of inclusion body formation was detected in the substantia nigra pars compacta, striatum, hippocampus, or selected regions of the cerebral cortex. We also noted tau/ubiquitin-immunoreactive aggregates in pigmented neurons of the substantia nigra pars compacta and locus coeruleus in all four DYT1 dystonia cases, but not in controls. This study supports the notion that DYT1 dystonia is associated with impaired protein handling and the nuclear envelope. The role of the pedunculopontine and cuneiform nuclei, and related brainstem brainstem structures, in mediating motor activity and controlling muscle tone suggests that alterations in these structures could underlie the pathophysiology of DYT1 dystonia [corrected]

Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to ‘synthetic’ state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of ‘synthetic’ VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro‐inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase‐2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.

BACKGROUND. The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the “vulnerable plaque,” necessitating an improved approach for predicting onset of symptoms.METHODS. We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies.RESULTS. Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study.CONCLUSION. The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery.FUNDING. UK: British Heart Foundation (BHF); King’s BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London in partnership with King’s College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in insideout submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169 -185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinoneacceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD ؉ . The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg 2؉ was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg 2؉ . After exposure to elevated temperature (37°C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg 2؉ . The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD ؉ , and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/ de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.In mammalian mitochondria NADH-ubiquinone oxidoreductase (Complex I, coupling Site 1, EC 1.6.99.3) functions as the main entry to the respiratory chain. The enzyme has an extremely complex structure being composed of more than 40 different subunits (1, 2). It contains multiple distinct redox components (FMN, a number of iron-sulfur clusters and tightly bound ubiquinones) operating in unknown sequence of the reactions coupled with vectorial translocation of protons from matrix to intermembraneous space. The functions of a vast majority of the enzyme subunits are not known. Most of the recent studies on Complex I and its simpler procaryotic counterparts (Type 1 NADH dehydrogenases) have focused on their structure (1-4), iron-sulfur clusters location (5, 6), possible mechanism of proton translocation (7-9), and the comparative molecular biology of the enzyme (10 -12).Very little is known about regulatory properties of Complex I. The bovine heart enzyme shows very complex kinetic behavior when assayed in either forward or reverse reactions. Following pioneering observations of Estabr...